Uniparental Genetic Heritage of Belarusians: Encounter of ...
Transcript of Uniparental Genetic Heritage of Belarusians: Encounter of ...
University of Huddersfield Repository
Kushniarevich, Alena, Sivitskaya, Larysa, Danilenko, Nina, Novogrodskii, Tadeush, Tsybovsky, Iosif, Kiseleva, Anna, Kotova, Svetlana, Chaubey, Gyaneshwer, Metspalu, Ene, Sahakyan, Hovhannes, Bahmanimehr, Ardeshir, Reidla, Maere, Rootsi, Siiri, Parik, Jüri, Reisberg, Tuuli, Achilli, Alessandro, Hooshiar Kashani, Baharak, Gandini, Francesca, Olivieri, Anna, Behar, Doron M., Torroni, Antonio, Davydenko, Oleg and Villems, Richard
Uniparental Genetic Heritage of Belarusians: Encounter of Rare Middle Eastern Matrilineages with a Central European Mitochondrial DNA Pool
Original Citation
Kushniarevich, Alena, Sivitskaya, Larysa, Danilenko, Nina, Novogrodskii, Tadeush, Tsybovsky, Iosif, Kiseleva, Anna, Kotova, Svetlana, Chaubey, Gyaneshwer, Metspalu, Ene, Sahakyan, Hovhannes, Bahmanimehr, Ardeshir, Reidla, Maere, Rootsi, Siiri, Parik, Jüri, Reisberg, Tuuli, Achilli, Alessandro, Hooshiar Kashani, Baharak, Gandini, Francesca, Olivieri, Anna, Behar, Doron M., Torroni, Antonio, Davydenko, Oleg and Villems, Richard (2013) Uniparental Genetic Heritage of Belarusians: Encounter of Rare Middle Eastern Matrilineages with a Central European Mitochondrial DNA Pool. PLoS ONE, 8 (6). e66499. ISSN 1932-6203
This version is available at http://eprints.hud.ac.uk/id/eprint/25744/
The University Repository is a digital collection of the research output of theUniversity, available on Open Access. Copyright and Moral Rights for the itemson this site are retained by the individual author and/or other copyright owners.Users may access full items free of charge; copies of full text items generallycan be reproduced, displayed or performed and given to third parties in anyformat or medium for personal research or study, educational or not-for-profitpurposes without prior permission or charge, provided:
• The authors, title and full bibliographic details is credited in any copy;• A hyperlink and/or URL is included for the original metadata page; and• The content is not changed in any way.
For more information, including our policy and submission procedure, pleasecontact the Repository Team at: [email protected].
http://eprints.hud.ac.uk/
Uniparental Genetic Heritage of Belarusians: Encounterof Rare Middle Eastern Matrilineages with a CentralEuropean Mitochondrial DNA PoolAlena Kushniarevich1*, Larysa Sivitskaya2, Nina Danilenko2, Tadeush Novogrodskii3, Iosif Tsybovsky4,
Anna Kiseleva4, Svetlana Kotova4, Gyaneshwer Chaubey1, Ene Metspalu5, Hovhannes Sahakyan1,6,
Ardeshir Bahmanimehr6, Maere Reidla5, Siiri Rootsi1, Juri Parik1,5, Tuuli Reisberg1,5, Alessandro Achilli7,
Baharak Hooshiar Kashani8, Francesca Gandini8, Anna Olivieri8, Doron M. Behar9, Antonio Torroni8,
Oleg Davydenko2, Richard Villems5,10
1 Estonian Biocentre, Tartu, Estonia, 2 Institute of Genetics and Cytology, National Academy of Sciences of Belarus, Minsk, Belarus, 3 Belarusian State University, Faculty of
History, Department of Ethnology, Museology and History of Arts, Minsk, Belarus, 4 Centre of Forensic Expertise and Criminalistics, Minsk, Belarus, 5 Department of
Evolutionary Biology, Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia, 6 Human Genetics Group, Institute of Molecular Biology, Academy of
Sciences of Armenia, Yerevan, Armenia, 7 Dipartimento di Biologia Cellulare e Ambientale, Universita di Perugia, Perugia, Italy, 8 Dipartimento di Biologia e Biotecnologie
‘‘L. Spallanzani’’, Universita di Pavia, Pavia, Italy, 9 Molecular Medicine Laboratory, Rambam Health Care Campus, Haifa, Israel, 10 Estonian Academy of Sciences, Tallinn,
Estonia
Abstract
Ethnic Belarusians make up more than 80% of the nine and half million people inhabiting the Republic of Belarus.Belarusians together with Ukrainians and Russians represent the East Slavic linguistic group, largest both in numbers andterritory, inhabiting East Europe alongside Baltic-, Finno-Permic- and Turkic-speaking people. Till date, only a limitednumber of low resolution genetic studies have been performed on this population. Therefore, with the phylogeographicanalysis of 565 Y-chromosomes and 267 mitochondrial DNAs from six well covered geographic sub-regions of Belarus westrove to complement the existing genetic profile of eastern Europeans. Our results reveal that around 80% of the paternalBelarusian gene pool is composed of R1a, I2a and N1c Y-chromosome haplogroups – a profile which is very similar to thetwo other eastern European populations – Ukrainians and Russians. The maternal Belarusian gene pool encompasses a fullrange of West Eurasian haplogroups and agrees well with the genetic structure of central-east European populations. Ourdata attest that latitudinal gradients characterize the variation of the uniparentally transmitted gene pools of modernBelarusians. In particular, the Y-chromosome reflects movements of people in central-east Europe, starting probably as earlyas the beginning of the Holocene. Furthermore, the matrilineal legacy of Belarusians retains two rare mitochondrial DNAhaplogroups, N1a3 and N3, whose phylogeographies were explored in detail after de novo sequencing of 20 and 13complete mitogenomes, respectively, from all over Eurasia. Our phylogeographic analyses reveal that two mitochondrialDNA lineages, N3 and N1a3, both of Middle Eastern origin, might mark distinct events of matrilineal gene flow to Europe:during the mid-Holocene period and around the Pleistocene-Holocene transition, respectively.
Citation: Kushniarevich A, Sivitskaya L, Danilenko N, Novogrodskii T, Tsybovsky I, et al. (2013) Uniparental Genetic Heritage of Belarusians: Encounter of RareMiddle Eastern Matrilineages with a Central European Mitochondrial DNA Pool. PLoS ONE 8(6): e66499. doi:10.1371/journal.pone.0066499
Editor: Toomas Kivisild, University of Cambridge, United Kingdom
Received January 31, 2013; Accepted May 6, 2013; Published June 13, 2013
Copyright: � 2013 Kushniarevich et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by the State Committee on Science and Technology of the Republic of Belarus (SCST), Progetti Ricerca Interesse Nazionale2009 (Italian Ministry of the University) (to AA and AT), FIRB-Futuro in Ricerca 2008 (Italian Ministry of the University) (to AA and AO), Fondazione Alma MaterTicinensis (to AT), Estonian Basic Research grant SF0182474 (to RV and EM), Estonian Science Foundation grants (7858) (to EM), European Commission grant(ECOGENE205419) (to RV), the European Union Regional Development Fund (through the Centre of Excellence in Genomics (to RV). The funders had no role instudy design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: Co-authors Alessandro Achilli and Doron M Behar are Academic Editors for PLOS ONE. This does not alter the authors’ adherence to allthe PLOS ONE policies on sharing data and materials.
* E-mail: [email protected]
Introduction
Contemporary Belarus occupies the central-western fringe of
the East European plain. Its current landscape is due to features
acquired from glacier activities, which finally retreated around 12
thousand years ago (kya) [1]. Starting from this period, the
Belarusian territory is thought to have been completely and
continuously populated by Anatomically Modern Humans
(AMH). However, the prehistoric period per se lasted longer since
the earliest evidence of AMH activities in the present territory of
Belarus are dated to the Middle Upper Paleolithic period (20–
25 kya) [2,3].
Human genome variation has been successfully used to
reconstruct the lengthy prehistory of human populations and
two genetic loci, in particular, the non-recombining portion of the
Y-chromosome (NRY) and mitochondrial DNA (mtDNA), have
proven to be very informative [4–7]. The uniparental gene pools
have been explored at different levels among eastern Europeans.
Previous studies have shown that the maternal gene pool in East
PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e66499
Europe is composed of typical West Eurasian haplogroups and is
characterized by very similar compositions among populations [8–
14]. Studies on Y-chromosome indicate the composite regional
background of paternal gene pools (e.g. R1a, I lineages) and the
substantial substructure of eastern European populations [15–29].
There are only a few studies performed at a low level of
molecular resolution and based on restricted sampling, which
target common NRY and mtDNA variation in Belarusians [30,31]
and infer the intra-population structure using Y-chromosome
Short Tandem Repeats (Y-STRs) [24,32]. Hence, comprehensive
profiling of the gene pool of Belarusians within central-east
Europeans along with possible sources of prevalent and rare
uniparental lineages has remained unexplored.
In this study, we aim to unravel the genetic structure of
Belarusians using high resolution analysis of 565 Y-chromosomes
and 267 mtDNAs representing six geographic sub-regions of
Belarus and to evaluate the temporal and geographic origin of
their most common and rare lineages. Furthermore, we studied in
detail the phylogeny and phylogeography of two common
Belarusian NRY lineages (N1c(Tat) and I2a(P37)) and, at the
level of complete mitogenomes, we investigated two deeply rooted
mtDNA haplogroups (N1a3 and N3), which are generally rare but
were observed in Belarusians and therefore are potentially
informative from the phylogeographic perspective.
Results and Discussion
Maternal gene pool of BelarusiansA total of 267 individuals from six geographic regions of Belarus
(Figure 1, Table S1) were included in the study of mtDNA
diversity. Fifty-eight sub-haplogroups, as well as paraphyletic
groups within them, were identified, all descending from the two
basal Eurasian mtDNA haplogroups M and N(R) (Figure 2, Table
S2).
Majority of the detected haplogroups are those diversified
primarily within Europe and those characterizing the central-east
European mtDNA pool. Among all H sub-branches identified,
H1b and H2a are well represented (Figure 2), which is in
agreement with previously reported studies of eastern European
populations [33,34]. Belarusian haplogroup V is characterized by
three first hypervariable segment (HVS-I) haplotypes (151–153 in
Table S2), which are more prevalent in East Europe than in West
Europe [11,26,35,36]. Similarly, U5a, another frequent hap-
logroup, is shown to be more typical for eastern Europeans
compared to central and south-eastern ones, whereas haplogroup
U5b reflects the input from south-west and Central Europe [37].
Certain Belarusian U4 sub-haplogroups, e.g. U4a2, are shown to
be well represented among other Slavic-speaking populations of
central-east Europe and to have expanded during the last 5–8 ky
[38]. Haplogroups J and T include those haplotypes that have
been shown to be European as well as Near-Eastern specific
(Figure 2) [39]. It was suggested that particular branches within J
and T were in Europe already during the Late Glacial period,
whereas only a few sub-lineages showed signs of Neolithic input
from the Near East [39].
Less frequent in Belarusians are mtDNA haplogroups stemming
directly from the N-node (except the R-branch), suggesting a
genetic contribution from Near/Middle East region. The N1a1
HVS-I motif (haplotype 95 in Table S2) detected in Belarusians
has been shown to be common among first farmers in Central
Europe, but rare in the preceding Paleolithic/Mesolithic popula-
tions and in modern Europeans [40,41]. Haplogroup I1a is
another lineage most likely associated with the Neolithic, whereas
haplogroup W seems to have expanded within Europe earlier [42].
N1a3 and N3 mtDNAs are also likely evidences of Middle Eastern
genetic traces in Europeans.
The smallest component of the Belarusian maternal gene pool
comprises of four East Asian specific M-rooted haplogroups
(Figure 2). It has been demonstrated that such a minor share of
East Eurasian mtDNAs is steady among western and central-east
Europeans irrespective of their linguistic affiliation, while it
increases notably eastward within East Europe reaching up to
one-third among particular Volga-Uralic populations [13,43].
To visualize the intra-population structure of Belarusians we
used principle component (PC) analysis based on haplogroup
frequencies. West and East Polesie (southern Belarus) are scattered
Figure 1. Geographic position of Belarus within Europe. Map of Belarus demonstrating the six geographic sub-regions studied is shown onthe right. Numbers 1–19 correspond to the location of sampling points (Table S1). Lit – Lithuania, Lat – Latvia, Est – Estonia.doi:10.1371/journal.pone.0066499.g001
Uniparental Genetic Heritage of Belarusians
PLOS ONE | www.plosone.org 2 June 2013 | Volume 8 | Issue 6 | e66499
in the PC plots and shifted from other sub-regions due to lower
frequencies of haplogroups J and U5 and higher frequencies of T,
H2 and K (PC plots PC1vsPC2 and PC1vsPC3 in Figure S1).
However, this could be, partly, due to events of genetic drift within
these relatively small regions. MtDNA haplogroups in general
demonstrate notable frequency patterns only in a wider geograph-
ic context, e.g. within West Eurasia [7,33,39], whereas within a
smaller region, such as the East European plain, these patterns are
not seen for all lineages (Table S3). We examined the genetic
variance between two major geographical subdivisions in Belar-
usians, in particular, southern vs the remaining four sub-
populations and western vs the remaining sub-groups (see the
Methods section for grouping details) using analysis of molecular
variance (AMOVA). Our results suggest that the genetic
differentiation between southern and the rest of the sub-groups
was marginally significant (Table S4) although the inter-group
variation was low (0.32%). In addition, pairwise Fst values tested
between six Belarusian sub-populations indicate very low inter-
population genetic differentiation (Table S5). Altogether, our
mtDNA analyses suggest that there is no strong evidence of
substructure within the Belarusian maternal gene pool.
Frequencies of Belarusian mtDNA haplogroups do not differ
considerably from other eastern European and Balkan popula-
tions, at least when major clades such as H1, H2, V, U5a and
U5b, K, T and J are considered (Table S3). However, populations
from the easternmost fringe of the eastern European region, the
Volga-Uralic, have a decreased share of overall H mtDNAs and a
noticeably increased frequency of haplogroup U4 as well as M-
lineages compared to Belarusians (Table S3). Our first PC analysis
based on mtDNA haplogroup frequencies revealed that Belar-
usians along with majority of other eastern European and Balkan
populations formed a single cluster while the members of the
Volga-Uralic region except Mordvins are separated from rest of
the studied populations with Udmurts and Bashkirs being the most
remote on the plot, as expected due to their distinctive genetic
compositions (Figure S2). Hence, to increase the resolution within
Figure 2. Phylogeny of mtDNA haplogroups and their relative frequencies in Belarusians. The tree is rooted relative to the RSRSaccording to [51]. Belarusian sub-populations are designated as BeE – East, BeWP – West Polesie, BeEP – East Polesie, BeN – North, BeC – Centre, BeW– West. Sample sizes and absolute frequencies are also given.doi:10.1371/journal.pone.0066499.g002
Uniparental Genetic Heritage of Belarusians
PLOS ONE | www.plosone.org 3 June 2013 | Volume 8 | Issue 6 | e66499
the former group, we performed the second PC analysis excluding
Bashkirs and Udmurts (Figure 3). In the resulting PC plot the
populations clustered more or less according to their geographic
affinities with Belarusians grouping together with their immediate
neighbors - Russians, Ukrainians, Poles and Lithuanians, and
being somewhat separated from Czechs and Slovaks as well as
representatives of the Balkan region (Figure 3).
Revision of the mtDNA N1a3 phylogenyTwo Belarusian mtDNAs, characterized by an unusual HVS-I
motif (A16240c, A16265G from the N-root), were classified as
members of N1a3 based on the A16265G transition (Table S2).
MtDNAs bearing A16265G along with C16201T relative to the
N-node were assigned to haplogroup N1a3 (ex-N1c) [44] for the
first time in [6] and since then this was considered as the diagnostic
motif for N1a3.
At the level of mtDNA control-region variation, the geographic
distribution of N1a3 is well described to date: it is primarily
confined to the Middle East, with the highest frequencies but a
rather low diversity, as far as HVS-I is concerned, in populations
of the Arabian Peninsula [42,45] and in the Marsh Arabs of Iraq
[46]. It is also present in the Caucasus region, most frequently
among Armenians, but also among Georgians, as well as in Adygei
and Dagestan people of the North Caucasus (our unpublished
data). N1a3 is found throughout the north-east of the Mediter-
ranean basin (Sicily, Rhodes, Crete, Cyprus, among Lebanese and
Palestinians) and in the Turkish Kurds ([47] and our unpublished
data). It is extremely rare in central-east Europe: single mtDNAs
have been found among Romanians, Poles, Belarusians and
among populations of the Volga-Ural region, Tatars and
Mordvins ([12,13,48,49] and our unpublished data). We note that
unusually high incidence of the N1a3 among Mordvins, living in
the East European plain of Russia, is meanwhile characterized by
low HVS-I diversity, indicative of a possible founder event in
recent times.
In contrast to the numerous HVS-I data, only a few complete
sequences characterize the phylogeny of haplogroup N1a3 [42].
Therefore, to extend our knowledge about the phylogeography of
N1a3, as well as to clarify the marker state of the C16201T
substitution, we completely sequenced two N1a3 mtDNAs (one
Belarusian and one Iranian Azeri), lacking the C16201T, along
with 18 N1a3 samples with the classical C16201T-A16265G motif
and originating from the Near/Middle East, North and South
Caucasus, South, Central and East Europe. These 20 novel
sequences were combined with eight mitogenomes published
previously [42,50,51] and the resulting phylogenetic tree is shown
in Figure 4.
The tree includes a major sub-branch defined by the C16201T
substitution and named here as N1a3a, which encompasses most
of the analyzed samples, whereas two haplotypes (Belarusian and
Iranian Azeri), form two individual twigs (Figure 4). Such
phylogenetic picture favors the overall stability of the C16201T
transition in the phylogeny of mtDNA (www.mtdnacommunity.
org and [44]), however, two independent back T16201C
mutations remain a possibility.
Our wide geographic coverage of N1a3 mtDNAs identifies
some features of this haplogroup. N1a3 mtDNAs from the Near/
Middle East and the Caucasus include diverse set of haplotypes
differing from those found in European populations. N1a3
mtDNAs from South Europe form individual limbs (Italian), but
some share substitutions with the Middle Eastern and Caucasian
N1a3 mtDNAs, while others – with mtDNA from central-east
Figure 3. PC analysis based on mtDNA haplogroup frequencies among eastern Europeans and Balkan populations. The contributionof each haplogroup to the first and the second PCs is shown in gray. The group ‘‘Other’’ includes ‘‘Other’’ from published data merged withuncommon haplogroups L1b, L2a and L3f. Frequencies of mtDNA haplogroups and references are listed in Table S3.doi:10.1371/journal.pone.0066499.g003
Uniparental Genetic Heritage of Belarusians
PLOS ONE | www.plosone.org 4 June 2013 | Volume 8 | Issue 6 | e66499
Figure 4. Maximum parsimony tree of mtDNA haplogroup N1a3. The tree includes 20 novel complete sequences (marked with an asteriskand underlined accession numbers) and eight previously published [42,50(and references therein),51]. Mutations relative to the RSRS [51] areindicated on the branches; transversions are specified with a lower case letter; Y and R stand for heteroplasmy; underlining indicates positionsexperiencing recurrent mutations within the tree while exclamation marks refer to one (!) or two (!!) back mutations relative to the RSRS. Coalescenceage estimates for N1a3 and N1a3a obtained by employing the complete genome and synonymous (r) clocks, indicated by # and @, respectively, arealso shown.doi:10.1371/journal.pone.0066499.g004
Uniparental Genetic Heritage of Belarusians
PLOS ONE | www.plosone.org 5 June 2013 | Volume 8 | Issue 6 | e66499
Europeans. N1a3 from central-east Europe encompasses both
highly divergent haplotypes (Romanian and Tatar) along with
almost ‘‘nodal-like’’ sequences (Mordvinian and Polish), charac-
terized only by single substitutions in HVS-II. Note that central-
east European N1a3 mtDNAs do not share substitutions with
those from the Near and Middle East (Figure 4).
It has been suggested earlier that the Near/Middle East is most
likely the region where haplogroup N1a3 has originated [42]. We
found that N1a3 mtDNAs in extant human populations coalesce
at 12–15 kya (Figure 4) and show distinct profiles in different
geographic regions. Our data suggest that the expansion of N1a3
bearers within the Near/Middle East, Caucasus and likely in
Europe took place during the Pleistocene-Holocene transition.
The lack of shared N1a3 haplotypes between Near/Middle East/
Caucasus and central-east Europe suggests absence of recent gene
flow between these regions. It is likely that N1a3 mtDNAs
experienced a period of diversification in all regions which might
have been enhanced also by the low number of N1a3 bearers
(either initially or decreased later).
Phylogeny and phylogeography of mtDNA haplogroupN3
One out of 267 Belarusian mtDNAs was a member of
haplogroup N3, a recently defined branch of the macro-
haplogroup N (www.mtdnacommunity.org and [44]). Haplogroup
N3 is characterized by the HVS-I motif T16086C, A16129G,
T16172C, T16217C, G16230A, T16278C, C16311T, C16519T
relative to the Reconstructed Sapiens Reference Sequence (RSRS)
[51] and thus far only two N3 mitogenomes of unknown
geographic origin have been reported [51]. The detection of a
new basal branch of macro-haplogroup N, in particular among
European populations, is a rare event, as its variation at the basal
level was comprehensively characterized for this region more than
a decade ago, when haplogroups R, X, I and W, which derive
from haplogroup N, were defined by a combined HVS-I/RFLP
(Restriction Fragment Length Polymorphism) approach [52].
Haplogroup N branches, typical of West Eurasia, have reached
complete genomic characterization by now [7,42] with four basal
lineages (N1, N2, R and X), among which all but haplogroup R
are, as a rule, infrequent, or, indeed, often very rare in Europe.
Other N basal branches have been found elsewhere in East and
South-East Asia, in Melanesia and Australia [53,54].
The analysis of more than 30 000 HVS-I sequences including
published and unpublished data shows that haplogroup N3 is
extremely rare in the global human population (Figure S3, Table
S6). Only 42 matching sequences have been found, suggesting an
overall frequency in West Eurasia around 0.1% and even less in
Europe (Figure S3). The majority of N3 mtDNAs are found in the
Middle East, in agreement with an ancestral homeland in that
area. N3 mtDNAs are not detected among about 2000 Africans
but one Egyptian from the current study, not in the Caucasus
(n = 2300), not across the Volga-Uralic region (n = 1200), Central
and East Asians (n = 3500), Siberians (n = 600) and Native
Americans. It was also not seen among more than 2000 subjects
of South Asia either (beside one Pathan-speaking individual from
Pakistan) (Figure S3, Table S6).
To get some insight into the phylogeny and phylogeography of
this novel haplogroup, we sequenced 13 N3 mitogenomes and
analyzed them together with three published mtDNAs [50,51].
The overall phylogeny of the 16 N3 mitogenomes is shown in
Figure 5.
Thirteen mtDNAs form a major star-like clade within the tree,
named N3a, which is defined by the motif T5048C-C9815T-
A11128G. This sub-branch includes the Belarusian mtDNA and
all European members of N3, together with several mtDNAs from
Iran. A second sub-branch of N3, defined also by three transitions
in the coding region (T5553C, C9211T, T15670C), encompasses
two mtDNAs from Iran. Finally, an additional Iranian sequence
forms an individual twig (Figure 5).
Considering 17 substitutions that separate the N-root from the
N3-root (Figure 5), haplogroup N3 has likely originated as early as
other N-branches. However, a very restricted number of its
descendants are detected in extant human populations. The
coalescence age of N3 mtDNAs points to an expansion at the
Pleistocene-Holocene boundary (12 kya with 95% confidence
intervals from 4 to 20 ky), whereas the major N3a sub-clade
expanded relatively recently, likely within the last 5000 years
(Figure 5).
The highest incidence and diversity of N3 mtDNAs are found in
populations of present-day Iran, indicating its territory as the most
likely ancestral homeland for the haplogroup. It appears that N3a
has spread ‘‘successfully’’ in the last millennia from the Iranian
area reaching North Africa in the south-west and central-east
Europe in the north-west, but not the Caucasus area or central-
south Asia. Taking into account the distribution pattern of N3 in
the Middle East extending west to the Balkan region up to
territories of Bulgaria and Romania, and its virtual absence
elsewhere, it is most likely that N3 has dispersed to Europe
through the Anatolian-Balkan path.
Although the spread patterns of haplogroups N1a3 and N3 bear
obvious similarities (Figures 4 and 5), it is worthwhile to notice
some of the differences. While the former is well spread all over the
western Asia – in Iran as well as in Arab-speaking areas and in the
Caucasus, the latter is largely restricted to Iran. It suggests that
movement of populations over the West Asian space during the
Pleistocene-Holocene boundary, coinciding perhaps with the end
of the Younger Dryas, was more intense than in later periods,
during the mid-Holocene, well after the transition of human to
agriculture and largely sedentary lifestyle.
NRY profile of BelarusiansMore than one half of Belarusian males belong to the
haplogroup R1a(SRY1532), which together with I2a(P37) and
N1c(Tat) NRY lineages cover almost 80% of the NRY diversity of
population; the rest is represented by numerous less frequent
haplogroups and sub-haplogroups (Figure 6). A substantial portion
of R1a chromosomes, bearing the derived M458G allele, have an
indigenous European origin [55]. In Belarusians, another branch
of haplogroup R, haplogroup R1b(M269), makes up around 5%
(Figure 6). It was shown previously [56] that this lineage
encompasses several sub-branches (i.e. East European-, Caucasus-
and south Siberian-specific) among eastern Europeans including
Belarusian population. Haplogroup I in Belarusians is composed
of multiple genetic inputs, mainly from the north-western Balkans
(I2a(P37)), and, to a lesser extent, from West and north-west
Europe (I1(M253), I2b(M223)) [57]. N1c(Tat) along with its much
less frequent sister group N1b(P43) (previously N2), detected in
Belarusians indicate an ancient patrilineal gene flow from the
north Eurasia westward, yet in the context of studied here
populations is best explained by partially shared Y-chromosomal
ancestry of Belarusians and their northern neighbors, Lithuanians
and Latvians, among whom N1c(Tat) reaches frequencies above
40% [20,21,26,58]. The STR haplotypes of N1b(P43) Y-
chromosomes belong to both ‘European’ and ‘Asian’ sub-clusters
[58], which may indicate their different sources in Belarusians
(Table S7), whereas in general we note that Belarusian population
represents the westernmost fringe of N1b(P43) haplogroup
distribution detected to date. J2(M172) and E1b1b1a(M78) NRY
Uniparental Genetic Heritage of Belarusians
PLOS ONE | www.plosone.org 6 June 2013 | Volume 8 | Issue 6 | e66499
haplogroups, found in Belarusians at low frequencies, are generally
typical for Anatolia and the southern Balkans [59,60] as well as for
the Caucasus region (G2a) [61,62].
Certain NRY haplogroups show gradient-like patterns in their
frequency distribution in Belarus. For example, haplogroup
I2a(P37) makes up a quarter of the Y-chromosome pool in the
south regions (West and East Polesie), but decreases northward, in
agreement with the earlier observed south-west – north-east
spread of this haplogroup [57]. Contrary to that, haplogroup
N1c(Tat) shows the highest frequency (around 15%) in north-west
Belarus and is decreasing southward, as it could be expected,
bearing in mind that among Lithuanians N1c(Tat) comprises close
to a half of their Y-chromosomes [21]. Haplogroup
R1a(SRY1532) has slightly lower frequencies in West and East
Polesie compared with the rest of Belarus. The share of
R1a1a7(M458) Y-chromosomes vice versa decreases from south-
west toward north and east of Belarus when Polesie is considered
as one region. To test patterns of spatial distribution of the three
major NRY haplogroups (N1c(Tat), I2a(P37) and R1a(SRY1532))
in Belarusians, spatial autocorrelation analysis was performed. The
correlograms show that N1c(Tat) and I2a(P37) haplogroup
frequency gradients within the Belarusian region are not
statistically significant likely due to a small number of points and
a rather small geographic area, whereas haplogroup
R1a(SRY1532) demonstrates no regular pattern (Figure S4).
However we note that both N1c(Tat) and I2a(P37) NRY
haplogroups demonstrate statistically significant south-north gra-
dients within a wider Eastern European area [15].
To evaluate the intra-population structure of the paternal gene
pool, a Multidimensional Scaling (MDS) analysis based on
pairwise Rst values calculated from 13 Y-STRs among six sub-
populations was performed (Figure S5). Our analysis revealed that
south-central Belarus (West, East Polesie and Centre sub-
populations) is separated from the north-western regions (BeN
and BeW) on the plot, whereas the East sub-population positions
apart. Similarly, pairwise Fst values, calculated from NRY
haplogroup frequencies, indicate that both West and East Polesie
(southern Belarus) are differentiated from other sub-regions except
the Centre (BeC) (Table S8). We applied AMOVA to test the
distribution of genetic variance for the same geographic subdivi-
sions of Belarusians used in case of mtDNA data (see the Methods
section for grouping details in AMOVA analysis). Analysis shows
that the genetic differentiation between southern and the rest of
the sub-groups is more informative (1.9% between group
variation, although statistically insignificant) in comparison to
western vs the rest of the sub-groups (Table S4). Taken together,
Figure 5. Maximum parsimony tree of mtDNA haplogroup N3. The tree includes 13 novel (marked with an asterisk and underlined accessionnumbers) and three previously published [50,51] complete sequences. Mutations relative to the RSRS [51] are shown on the branches; transversionsare specified with a lower case letter; underlining indicates positions which experienced recurrent mutations within the tree, while the exclamationmark (!) refers to one back mutation relative to the RSRS. Rho coalescence time estimates and their confidence intervals for haplogroup N3 and itsmajor sub-branch N3a obtained from the complete genome clock are also shown.doi:10.1371/journal.pone.0066499.g005
Uniparental Genetic Heritage of Belarusians
PLOS ONE | www.plosone.org 7 June 2013 | Volume 8 | Issue 6 | e66499
differences of NRY gene pool within Belarus are more
pronounced along its south-to-north axis than between its western
and eastern regions. It has been shown that the same trend of
south-north differentiation of the paternal gene pool extends
eastward, encompassing sub-populations of the so-called ‘‘histor-
ical Russian area’’ [15], whereas it becomes less notable or even
transforms into a west-east distinction further westward [23].
Similarly to other East Slavic-speakers, almost a third of the
Belarusian paternal gene pool is constituted of two haplogroups,
I2a(P37) and N1c(Tat). The first indicates gene flow from south-
east Europe northward [57], while N1c(Tat) is largely spread
among north Eurasians and within central-east Europe reaches the
Ukrainians in the south, though only marginally, the Poles in the
west [58]. To determine the relationship of Y-STR haplotypes
between the populations of East Europe and the Balkans and also
to get an idea about the origin of the two haplogroups in the extant
Belarusian population, we have calculated their Median-Joining
(MJ) networks (Table S9).
Maximum Parsimony (MP) tree (based on MJ network) of
haplogroup N1c(Tat) includes West and East Slavic-speakers,
Balts, Estonians, Finns (from the south and north Karelian regions
of Finland) and populations of the Volga-Uralic region (Komis,
Udmurts, Maris, Chuvashes and Bashkirs) (Figure 7). N1c(Tat)
haplotypes tend to show regional specificity within East Europe.
Three groups of haplotypes can be distinguished based on their
prevalence among certain populations: the ones, most common
among the Volga-Uralic populations; those spread primarily
among Finns and N1c(Tat) haplotypes that are found largely
among Balts, and Slavic-speakers. It has been suggested that the
differentiation of N1c(Tat) haplotypes between Balts and the
Volga-Uralic populations was due to the splitting of a founder on
the way of its migration towards the Baltic Sea region and was
strengthened by genetic drift [20,22]. The N1c(Tat) tree in this
study indicates that Belarusians share a considerable portion of
haplotypes with Balts pointing to a shared patrilineal founder(s)
and history. Beside this, Belarusian N1c(Tat) encompasses
haplotypes both individual ones and those, shared with Finns as
well as with the Volga-Uralic populations (Figure 7). Hence, it is
possible that in addition to the suggested split of the N1c(Tat)
founder(s) during its spread westward, the diversity of haplogroup
N1c(Tat) as observed in Belarusians, could have been shaped by
reciprocal movements of its bearers within East Europe.
Unlike haplogroup N1c(Tat), the microsatellite haplotypes of
haplogroup I2a(P37) of Balts, East, West Slavic populations and
Balkan peoples (Bosnians, Croats and Slovenians) show a star-like
branching pattern (Figure 8). Furthermore, most of the I2a(P37)-
haplotypes analyzed are shared among populations inhabiting a
wide geographic area.
Thus, the analyses reveal similar I2a(P37)-founder(s) for
Belarusians and Balkan populations, whereas haplogroup
N1c(Tat) in Belarusians is an assemblage of largely ‘‘Balto-Slavic’’
specific haplotypes along with those spread in Volga-Uralic and
Finnic populations.
The PC plot based on the frequencies of NRY haplogroups
(Figure 9) assesses the relationships between paternal gene pools of
Belarusians and other eastern European and Balkan populations
speaking Slavic, Baltic, Finno-Permic and Turkic languages.
Belarusians, Russians and Ukrainians, the three East Slavic-
speakers and geographic neighbors, are the closest according to
their patrilineal legacy. We also note that southern sub-popula-
tions of Belarus group with Ukrainians whereas northern and
western ones are moved toward Volga-Uralic populations
(Figure 9) - the intra-population pattern that was observed also
in the MDS plot based on Y-STRs (Figure S5). Two West Slavic-
speaking populations (Czechs and Poles) are shifted from East
Slavs due to higher frequencies of R1b and R1a NRY
haplogroups, respectively (Table S3), whereas Slovaks remain
close to Eastern Slavic group. Among the South Slavic-speakers,
Slovenians and Croatians stay closer to East Slavic-speakers, while
Bosnians, Macedonians and Serbians form a distant group, mainly
due to high frequencies of haplogroups I2a(P37) and E (Table S3).
Balts, Estonians and Volga-Uralic populations, who are northern
Figure 6. Phylogeny of NRY haplogroups and their relative frequencies in Belarusians. Haplogroup-defining biallelic markers are inparentheses. Belarusian sub-populations are designated as BeN – North, BeC – Centre, BeE – East, BeW – West, BeWP – West Polesie, BeEP – EastPolesie. Sample sizes and absolute frequencies are also given.doi:10.1371/journal.pone.0066499.g006
Uniparental Genetic Heritage of Belarusians
PLOS ONE | www.plosone.org 8 June 2013 | Volume 8 | Issue 6 | e66499
and eastern neighbors of the Slavic-speakers, stay also apart due to
considerably different frequencies of NRY haplogroups observed
in East Slavic-speakers, that is, a prevalence of haplogroup
N1c(Tat), a decreased R1a(SRY1532) and minute frequencies of
I2a(P37) (Figure 9, Table S3). Hereby, PC analysis reveals that the
NRY pool of central and eastern Europeans and Balkan
populations harbors a marked geographic structure, whereas
Belarusians, Ukrainians and Russians (except northern regions of
the latter) tend to cluster in agreement with their common East
Slavic linguistic affiliation and previously published observations
[15].
The intra-population structuring of BelarusiansThe watershed between the Baltic Sea and the Black Sea divides
Belarus roughly into north-eastern (rivers descending towards the
Baltic Sea basin) and south-western (rivers descending towards the
Black Sea) regions. The latter, known as Polesie, is vast lowland
rich in swamps and forests, and differs markedly from the northern
region of Belarus. Rural populations all over Belarus, non-uniform
in physical characteristics, speak numerous dialects, as an outcome
of a long-lasting sedentary agricultural lifestyle [63–65]. Yet the
pattern of their genetic variation does not follow only isolation-by-
distance differentiation caused by random genetic drift. As it was
Figure 7. Maximum Parsimony tree (based on MJ network) of NRY haplogroup N1c(Tat) calculated from seven Y-STRs. Volga-Uralicpopulations include Komis (Priluzhski, Izhevski), Udmurts, Maris, Bashkirs, Chuvashes. Altogether 402 individuals are analyzed, the sample size of eachpopulation and the set of Y-STRs used for calculations are given in Table S9.doi:10.1371/journal.pone.0066499.g007
Figure 8. Maximum Parsimony tree (based on MJ network) ofNRY haplogroup I2a(P37) calculated from seven Y-STRs. Balkanpopulations include Bosnians, Croatians and Slovenians. Altogether 347individuals are analyzed, the sample size of each population and the setof Y-STRs used for calculations are given in Table S9.doi:10.1371/journal.pone.0066499.g008
Uniparental Genetic Heritage of Belarusians
PLOS ONE | www.plosone.org 9 June 2013 | Volume 8 | Issue 6 | e66499
pointed above, these largely latitudinal gradients within the
Belarus people, reflect likely ancient movements of Y-chromo-
somes (males) from north to south, as testified by the spread
pattern of N1c(Tat)-chromosomes with their frequency peak
around 50–70% in eastern Fennoscandia [26,27,58], and,
secondly, the northward movements of the carriers of NRY
haplogroup I2a(P37), that have likely originated from north-
western Balkans [57,60]. The longitudinal Y-chromosomal intra-
Belarus variation is less pronounced (Table S4) likely because the
present-day Belarus area lies within the very epicenter of the initial
spread of the dominant R1a (among the Belarus population
haplogroup), including its R1a1a7(M458) limb, around the
Pleistocene-Holocene boundary [55]. Because the spread of
I2a(P37) Y-chromosomes from the Balkans, as well as the
expansion of N1c(Tat) in East Europe, have been dated to early
Holocene [26,60], one may conclude that the core of the
Belarusian patrilineal pool, comprising about three quarters of
its present-day variation, may have been formed during the post-
Younger Dryas - early Holocene period.
Our mtDNA data, on the other hand, suggest that genetic
variation between sub-populations in Belarusians is low. Whereas
PC and AMOVA analyses indicate a slight difference between
southern Belarusians and the rest of the sub-populations (Figure
S1, Table S4), pairwise population Fst values reveal very little or
no genetic differentiation (Table S5). Thereby, much larger and
deeper (preferentially complete mtDNA genomic level) studies of
Belarusians and their neighboring populations would be needed to
reliably reveal the potential shared ancestry and matrilineal gene
flows in the region.
It is also worth noting that high-density whole genome studies
[66] show that the genetic structure of Belarusians, similarly to that
of their immediate neighbors, largely comprises of two major
ancestry components spread across the north-eastern and southern
European regions with marginal East Eurasian-specific contribu-
tion.
To sum up, the phylogeography of the Belarusian patrilineal
heritage, although overwhelmingly West Eurasian by descent and
dominated by R1a, the most prevalent haplogroup among West
Figure 9. PC plot based on NRY haplogroup frequencies among eastern Europeans and Balkan populations. The contribution of eachhaplogroup to the first and the second PCs are shown in gray. Population abbreviations are as follows: BeN, BeW, BeC, BeWP, BeEP, BeE – Belarusiansfrom North, West, Central, West Polesie, East Polesie and East sub-regions, respectively, filled red circle denotes the total Belarusian population; RuS,RuC, RuN – Russians from southern, central and northern regions, respectively; Finns K – Finns from Karelia. K*(x N,P) refers to samples with M9, M20,M70 derived alleles and 92R7, M214 ancestral alleles; P*(xR) refers to samples with 92R7, M242 derived alleles and M207 ancestral allele; F*(xI,J,K)refers to samples with M89 derived allele and M9, M201, M170, 12f2 ancestral alleles; C(xF)DE refers to samples with Yap and M130 derived and M89ancestral alleles. Frequencies of NRY haplogroups and references are listed in Table S3.doi:10.1371/journal.pone.0066499.g009
Uniparental Genetic Heritage of Belarusians
PLOS ONE | www.plosone.org 10 June 2013 | Volume 8 | Issue 6 | e66499
and East Slavs, reveals two latitudinal gradients, reflecting
prehistoric and historic time admixture with the Baltic-speaking
people in the north (haplogroup N1c) and gene flow from the
north-western Balkans (haplogroup I2a). Meanwhile, East Eur-
asian Y-chromosomal (Q) and mitochondrial DNA (M including
C, D, G) variants are very rare among Belarusians. Detecting a
new basal branch of a macro-haplogroup N – haplogroup N3 –
among Belarusians, came as a surprise and provoked its further
study, alongside with somewhat less, but still rare haplogroup
N1a3. Mainly Middle Eastern, the phylogeography of haplogroup
N3 may represent a detectable gene flow from Middle East to
Europe during mid-Holocene. In contrast to N3, the phylogeog-
raphy of haplogroup N1a3 and its major sub-clade N1a3a,
particularly, demonstrates a high diversity over a wide geographic
area covering Middle East, Caucasus and Europe, and attests a
significantly earlier expansion of its bearers, likely, around the
Pleistocene-Holocene transition.
Methods
SamplesFor sampling reasons the territory of Belarus was divided into
six geographic sub-regions: North (West Dvina River region), East
(Dnieper River region), West (Neman River region), West Polesie
(south-west region of Belarus), East Polesie (south-eastern region of
Belarus) and Centre, the latter located in between the other five
(Figure 1). To avoid demographic effects due to the last century
industrial urbanization, sampling was carried out only in small
towns and villages. Altogether 565 Y-chromosomes and 267
mtDNAs of ethnic Belarusians were analyzed. The regions and
sample sizes are listed in Table S1.
All volunteers filled a detailed questionnaire ascertaining the
ethnicity and birth-place of themselves as well as their parents and
grandparents. Only adult volunteers, who resided in the region of
interest and whose ancestors had lived there for the last three
generations, were included in the study. Intra-venous blood was
collected from healthy unrelated males. DNA samples were
obtained using the standard protocol with proteinase K following
phenol-chloroform extraction and ethanol precipitation [67].
Ethics StatementThe population samples analyzed in the study were collected
after having obtained a written informed consent. The study has
been considered and approved specifically by the Bioethics
Committee of the Belarusian State Medical University (Minsk,
Belarus) and Scientific Boards of the participating research
institutions.
GenotypingY-chromosome data were generated by genotyping (RFLP or
direct sequencing) 28 single nucleotide polymorphisms (SNPs) and
indels (insertions, deletions) (M89, Yap, M35, M78, M123, M201,
P15, M170, M253, P37, M223, 12f2/SRY, M267, M172, M9,
M70, M231, P43, Tat, 92R7, M207, M173, SRY1532, M458,
M73, M269, M124, M242) in 565 samples according to the
current Y-chromosome phylogeny [55,68]. Note, the following
markers: M174 (haplogroup D), M130 (haplogroup C), M81
(within haplogroup E), M22 (haplogroup L) and M82 (haplogroup
H) were typed but not observed. In total, 17 NRY haplogroups
were inferred whereas two samples remained in a paragroup (F*(x
I, J, G, H, K)). Additionally, 14 Y-STRs were genotyped in all
samples (DYS19, DYS385ab, DYS389I,II, DYS390, DYS391,
DYS392, DYS393, DYS437, DYS438, DYS439, DYS448,
DYS456, DYS458 and H4).
For mtDNA, HVS-I from nucleotide positions 16000 to 16400
was sequenced in 267 Belarusian samples. Complete mtDNA
sequencing was performed for 33 samples in total, in part
according to [69], in part applying the methodology described in
[70]. Sequences were aligned and analyzed by using ChromasPro
version 1.5 (Technelysium Pty Ltd), and nucleotide mutations
were initially ascertained relative to the revised Cambridge
Reference Sequence (rCRS) [71]. Then, in order to record
HVS-I and complete mitogenome polymorphic positions relative
to the RSRS [51], the FASTmtDNA utility provided by MtDNA
Community (www.mtdnacommunity.org) was applied. HVS-I and
coding-region substitutions (Table S10) were used to resolve
haplogroup status following the hierarchy of the mtDNA
phylogenetic tree (www.mtdnacommunity.org and [44]). MtDNA
haplogroups were designated according to the current nomencla-
ture; transitions, transversions, back mutations were labeled
following the established style (www.mtdnacommunity.org and
[44]). Polymorphic nucleotide positions recorded relative to the
RSRS and rCRS for 33 completely sequenced mtDNAs in this
study are listed in Table S11.
Data analysisY-STR haplotype phylogenies for major NRY haplogroups in
the Belarusian population were constructed using Network 4.6.0.0,
applying the MJ algorithm (Fluxus Technology Ltd, http://fluxus-
technology.com). Weights of loci were chosen according to their
variability, post-processing MP calculations were performed and
MP trees of NRY haplogroups were drawn using Network
Publisher [72]. DYS385 was excluded from all further calcula-
tions, DYS389I was subtracted from DYS389II and both were
included in the calculations. When data from reference popula-
tions were included in the analysis of NRY haplogroup
phylogenies, the restricted available set of Y-STRs was used
(specified for each haplogroup in Table S9). The Y-STR
haplotypes for the N1c(Tat), N1b(P43) and I2a(P37) NRY
haplogroups of Belarusians are listed in Table S7 and Table
S12, respectively.
Arlequin 3.5 software [73] was used to calculate genetic distance
indices (Rst, Fst) and to assess the genetic structure in Belarusians
by AMOVA. Two major geographical subdivisions of Belarusians
were considered in AMOVA: (a) southern (West and East Polesie)
vs the remaining four sub-populations (Centre, West, East and
North) and (b) western (West and West Polesie) vs the remaining
four sub-populations (Centre, East Polesie, East and North). MDS
was performed using Statistica 6.0 Software (http://www.statsoft.
com). PC analysis was performed using the popstr algorithm
(http://harpending.humanevo.utah.edu/popstr/). Frequencies of
mtDNA and NRY haplogroups used in the PC analyses are listed
in Table S3. To test patterns of spatial distribution of the three
major NRY haplogroups in Belarusians (N1c(Tat), I2a(P37) and
R1a(SRY1532)), Moran’s I autocorrelation coefficients were
calculated using binary weight matrix with five distance classes
and random distribution assumption using in the PASSAGE
software V.1.1 (release 3.4) (http://www.passagesoftware.net/)
[74]. Note that six Belarusian sub-populations (Table S1) together
with immediate neighbors (Poles, Lithuanians, Latvians, Central
Russians and Ukrainians) were included in the analysis.
To estimate the age of mtDNA lineages, we calculated rho-
statistics (r) as average number of substitutions from the root
haplotype [75] and its standard deviation (s) [76]. The calculator
provided by [77] was used to convert the r-statistics and its error
ranges to age estimates with 95% confidence intervals.
Uniparental Genetic Heritage of Belarusians
PLOS ONE | www.plosone.org 11 June 2013 | Volume 8 | Issue 6 | e66499
Supporting Information
Figure S1 PC analysis based on mtDNA haplogroupfrequencies in six Belarusian sub-populations. The
distribution of the populations within 1–2 and 1–3 PCs is
represented in the upper panels; the contribution of mtDNA
haplogroups to each of the PCs is depicted in the lower panels. Sub-
populations are designated as BeN – North, BeC – Centre, BeE –
East, BeW – West, BeWP – West Polesie, BeEP – East Polesie.
(DOCX)
Figure S2 PC analysis based on mtDNA haplogroupfrequencies among eastern Europeans and Balkanpopulations. The contribution of each haplogroup to the first
and the second PCs is shown in gray. The group ‘‘Other’’ includes
‘‘Other’’ from published data merged with uncommon hap-
logroups L1b, L2a and L3f. Frequencies of mtDNA haplogroups
and references are listed in Table S3.
(DOCX)
Figure S3 The distribution of mtDNA haplogroup N3 inworld populations retrieved from published data alongwith those generated in this study. Black squares refer to the
screened population data; green circles mark regions where
haplogroup N3 has been detected and a star sign denotes the
geographic origin of N3 mtDNAs completely sequenced in this
study. Numbers inside the star correspond to the number of
mtDNAs. See Table S6 for reference data and number of N3
sequences detected in each population.
(DOCX)
Figure S4 Spatial autocorrelation analysis for threemajor NRY haplogroups (N1c(Tat), I2a(P37) andR1a(SRY1532)) in Belarusians. Moran’s I indices were
calculated for three NRY haplogroups in six Belarusian sub-
populations including also immediate neighbor populations
(Ukraine, Poland, Lithuania, Latvia, Central Russia). Correlo-
grams indicate that ‘gradient-like’ frequency patterns for N1c(Tat)
and I2a(P37) haplogroups are not statistically supported due to
likely small number of points and rather small geographic area.
Haplogroup R1a(SRY1532) demonstrates no pattern in its
frequency distribution. Open circles in correlograms denote non-
significant values.
(DOCX)
Figure S5 MDS plot of pair-wise Rst values obtainedfrom 13 Y-STRs in the six Belarusian sub-populations(stress = 0.0000048). Sub-populations are designated as follows:
BeN – North, BeC – Centre, BeE – East, BeW – West, BeWP –
West Polesie, BeEP – East Polesie.
(DOCX)
Table S1 Geographic origin of the Belarusian samples.(XLSX)
Table S2 MtDNA control and coding region polymor-phisms in Belarusians.(XLSX)
Table S3 MtDNA and NRY reference data used in PCanalyses.
(XLS)
Table S4 Analysis of molecular variance in Belarusians.
(DOCX)
Table S5 Pairwise population Fst calculated frommtDNA haplogroup frequencies in six Belarusian sub-populations.
(DOCX)
Table S6 The distribution of N3 mtDNAs (T16086C,A16129G, T16172C, T16217C, G16230A, T16278C,C16311T) in world populations.
(XLSX)
Table S7 Y-chromosome N1c(Tat) and N1b(P43) STRhaplotypes in Belarusians.
(XLSX)
Table S8 Pairwise population Fst calculated from NRYhaplogroup frequencies in six Belarusian sub-popula-tions.
(DOCX)
Table S9 Y-STR data used in Median-Joining Networkscalculations.
(XLSX)
Table S10 MtDNA haplogroups defining control (HVS-I:16000–16400) and coding-region mutations relative tothe RSRS and rCRS.
(XLSX)
Table S11 Complete haplotypes of N3 and N1a3mtDNAs generated in this study.
(XLSX)
Table S12 Y-chromosome I2a(P37) STR haplotypes inBelarusians.
(XLSX)
Acknowledgments
We thank all the volunteers who donated their blood and made this study
possible, and also all expedition members who participated in interviewing
and sampling. We thank M. Jarve for her valuable contribution to the
manuscript. We thank also two anonymous reviewers for their critical
comments on the manuscript.
Author Contributions
Conceived and designed the experiments: OD RV. Performed the
experiments: AK. Analyzed the data: AK AnK AO FG AA BHK.
Contributed reagents/materials/analysis tools: LS ND TN SK IT HS AB
EM JP TR SR MR. Wrote the paper: AK. Discussed the results and
commented on the manuscript: AK OG IT GC SR MR DMB AA AO AT
RV.
References
1. Mahnach AS, Garetski RG, Matveev AV (2001) Geologiya Belarusi. Minsk:
IGN NAN Belarusi. 815 p. (in Russian)
2. Svezhentsev YS, Popov SG (1993) Late Paleolithic chronology of the East
European Plain. Radiocarbon 35:495–501.
3. Zaikouski EM, Isaenka UF, Kalechyts AG, Kapycin VF, Kryvaltsevich MM
(1997) Arhealogiya Belarusi. Minsk: Belaruskaya Navuka. Vol. I. pp. 21–88. (in
Belarusian)
4. Rosser ZH, Zerjal T, Hurles ME, Adojaan M, Alavantic D, et al. (2000) Y-
chromosomal diversity in Europe is clinal and influenced primarily by
geography, rather than by language. Am J Hum Genet 67: 1526–1543.
5. Semino O, Passarino G, Oefner PJ, Lin AA, Arbuzova S, et al. (2000) The
genetic legacy of Paleolithic Homo sapiens sapiens in extant Europeans: a Y
chromosome perspective. Science 290: 1155–1159.
6. Richards M, Macaulay V, Hickey E, Vega E, Sykes B, et al. (2000) Tracing
European founder lineages in the Near Eastern mtDNA pool. Am J Hum Genet
67: 1251–1276.
Uniparental Genetic Heritage of Belarusians
PLOS ONE | www.plosone.org 12 June 2013 | Volume 8 | Issue 6 | e66499
7. Torroni A, Achilli A, Macaulay V, Richards M, Bandelt H-J (2006) Harvesting
the fruit of the human mtDNA tree. Trends Genet 22: 339–345.
8. Malyarchuk BA, Denisova GA, Derenko MV, Rogaev EI, Vlasenko LV, et al.
(2001) Variability in mitochondrial DNA in Russian inhabitants from Krasnodar
Krai, Belgorod and the lower Novgorod region. Genetika 37: 1411–1416.
9. Malyarchuk BA, Grzybowski T, Derenko MV, Czarny J, Wozniak M, et al.
(2002) Mitochondrial DNA variability in Poles and Russians. Ann Hum Genet
66: 261–283.
10. Kasperaviciute D, Kucinskas V (2002) Variability of the human mitochondrial
DNA control region sequences in the Lithuanian population. J Appl Genet 43:
255–260.
11. Pliss L, Tambets K, Loogvali E-L, Pronina N, Lazdins M, et al. (2006)
Mitochondrial DNA portrait of Latvians: towards the understanding of the
genetic structure of Baltic-speaking populations. Ann Hum Genet 70: 439–458.
12. Grzybowski T, Malyarchuk BA, Derenko MV, Perkova MA, Bednarek J, et al.
(2007) Complex interactions of the Eastern and Western Slavic populations with
other European groups as revealed by mitochondrial DNA analysis. Forensic Sci
Int Genet 1: 141–147.
13. Bermisheva M, Tambets K, Villems R, Khusnutdinova E (2002) Diversity of
mitochondrial DNA haplotypes in ethnic populations of the Volga-Ural region
of Russia. Mol Biol (Mosk) 36: 990–1001.
14. Morozova I, Evsyukov A, Kon’kov A, Grosheva A, Zhukova O, et al. (2012)
Russian ethnic history inferred from mitochondrial DNA diversity. Am J Phys
Anthropol 147: 341–351.
15. Balanovsky O, Rootsi S, Pshenichnov A, Kivisild T, Churnosov M, et al. (2008)
Two sources of the Russian patrilineal heritage in their Eurasian context.
Am J Hum Genet 82: 236–250.
16. Roewer L, Willuweit S, Kruger C, Nagy M, Rychkov S, et al. (2008) Analysis of
Y chromosome STR haplotypes in the European part of Russia reveals high
diversities but non-significant genetic distances between populations. Int J Legal
Med 122: 219–223.
17. Fechner A, Quinque D, Rychkov S, Morozowa I, Naumova O, et al. (2008)
Boundaries and clines in the West Eurasian Y-chromosome landscape: insights
from the European part of Russia. Am J Phys Anthropol 137: 41–47.
18. Bellusci G, Blasi P, Vershubsky G, Suvorov A, Novelletto A, et al. (2010) The
landscape of Y chromosome polymorphisms in Russia. Ann Hum Biol 37: 367–
384.
19. Khar’kov VN, Stepanov VA, Borinskaia SA, Kozhekbaeva ZM, Gusar VA, et
al. (2004) Structure of the gene pool of eastern Ukrainians from Y-chromosome
haplogroups. Genetika 40: 415–421.
20. Zerjal T, Beckman L, Beckman G, Mikelsaar AV, Krumina A, et al. (2001)
Geographical, linguistic, and cultural influences on genetic diversity: Y-
chromosomal distribution in Northern European populations. Mol Biol Evol
18: 1077–1087.
21. Kasperaviciute D, Kucinskas V, Stoneking M (2004) Y chromosome and
mitochondrial DNA variation in Lithuanians. Ann Hum Genet 68: 438–452.
22. Lappalainen T, Hannelius U, Salmela E, Von Dobeln U, Lindgren CM, et al.
(2009) Population structure in contemporary Swede - a Y-chromosomal and
mitochondrial DNA analysis. Ann Hum Genet 73: 61–73.
23. Kayser M, Lao O, Anslinger K, Augustin C, Bargel G, et al. (2005) Significant
genetic differentiation between Poland and Germany follows present-day
political borders, as revealed by Y-chromosome analysis. Hum Genet 117:
428–443.
24. Rebała K, Mikulich AI, Tsybovsky IS, Sivakova D, Dzupinkova Z, et al. (2007)
Y-STR variation among Slavs: evidence for the Slavic homeland in the middle
Dnieper basin. J Hum Genet 52: 406–414.
25. Wozniak M, Malyarchuk B, Derenko M, Vanecek T, Lazur J, et al. (2010)
Similarities and distinctions in Y chromosome gene pool of Western Slavs.
Am J Phys Anthropol 142: 540–548.
26. Tambets K, Rootsi S, Kivisild T, Help H, Serk P, et al. (2004) The western and
eastern roots of the Saami - the story of genetic ‘‘outliers’’ told by mitochondrial
DNA and Y chromosomes. Am J Hum Genet 74: 661–682.
27. Lappalainen T, Koivumaki S, Salmela E, Huoponen K, Sistonen P, et al. (2006)
Regional differences among the Finns: a Y-chromosomal perspective. Gene 376:
207–215.
28. Mirabal S, Regueiro M, Cadenas AM, Cavalli-Sforza LL, Underhill PA, et al.
(2009) Y-chromosome distribution within the geo-linguistic landscape of
northwestern Russia. Eur J Hum Genet 17: 1260–1273.
29. Mielnik-Sikorska M, Daca P, Wozniak M, Malyarchuk BA, Bednarek J, et al.
(2013) Genetic data from Y chromosome STR and SNP loci in Ukrainian
population. Forensic Sci Int Genet 7(1): 200–203.
30. Belyaeva O, Bermisheva M, Khrunin A, Slominsky P, Bebyakova N, et al. (2003)
Mitochondrial DNA variations in Russian and Belorussian populations. Hum
Biol 75: 647–660.
31. Khar’kov VN, Stepanov VA, Feshchenko SP, Borinskaia SA, Iankovskii NK, et
al. (2005) Frequencies of Y chromosome binary haplogroups in Belarussians.
Genetika 41: 1132–1136.
32. Rebała K, Tsybovsky IS, Bogacheva AV, Kotova SA, Mikulich AI, et al. (2011)
Forensic analysis of polymorphism and regional stratification of Y-chromosomal
microsatellites in Belarus. Forensic Sci Int Genet 5: e17–20.
33. Loogvali E-L, Roostalu U, Malyarchuk BA, Derenko MV, Kivisild T, et al.
(2004) Disuniting uniformity: a pied cladistic canvas of mtDNA haplogroup H in
Eurasia. Mol Biol Evol 21: 2012–2021.
34. Roostalu U, Kutuev I, Loogvali E-L, Metspalu E, Tambets K, et al. (2007)Origin and expansion of haplogroup H, the dominant human mitochondrial
DNA lineage in West Eurasia: the Near Eastern and Caucasian perspective. Mol
Biol Evol 24: 436–448.
35. Torroni A, Bandelt HJ, Macaulay V, Richards M, Cruciani F, et al. (2001) A
signal, from human mtDNA, of postglacial recolonization in Europe. Am J Hum
Genet 69: 844–852.
36. Malyarchuk BA, Derenko MV, Grzybowski T, Czarny J, Miscicka-Slivka D, et
al. (2002) Mitochondrial DNA variation in Russian populations of Stavropolkrai, Orel and Saratov oblasts. Genetika 38: 1532–1538.
37. Malyarchuk B, Derenko M, Grzybowski T, Perkova M, Rogalla U, et al. (2010)
The peopling of Europe from the mitochondrial haplogroup U5 perspective.PLoS ONE 5: e10285.
38. Malyarchuk B, Grzybowski T, Derenko M, Perkova M, Vanecek T, et al. (2008)
Mitochondrial DNA phylogeny in Eastern and Western Slavs. Mol Biol Evol 25:1651–1658.
39. Pala M, Olivieri A, Achilli A, Accetturo M, Metspalu E, et al. (2012)
Mitochondrial DNA signals of late glacial recolonization of Europe from neareastern refugia. Am J Hum Genet 90: 915–924.
40. Haak W, Forster P, Bramanti B, Matsumura S, Brandt G, et al. (2005) Ancient
DNA from the first European farmers in 7500-year-old Neolithic sites. Science310: 1016–1018.
41. Bramanti B, Thomas MG, Haak W, Unterlaender M, Jores P, et al. (2009)
Genetic discontinuity between local hunter-gatherers and central Europe’s firstfarmers. Science 326: 137–140.
42. Fernandes V, Alshamali F, Alves M, Costa MD, Pereira JB, et al. (2012) The
Arabian cradle: mitochondrial relicts of the first steps along the southern routeout of Africa. Am J Hum Genet 90: 347–355.
43. Malyarchuk BA, Perkova MA, Derenko MV (2008) Origin of the Mongoloid
component in the mitochondrial gene pool of Slavs. Genetika 44: 401–406.
44. Van Oven M, Kayser M (2009) Updated comprehensive phylogenetic tree of
global human mitochondrial DNA variation. Hum Mutat 30: E386–394.
45. Abu-Amero KK, Larruga JM, Cabrera VM, Gonzalez AM (2008) Mitochon-drial DNA structure in the Arabian Peninsula. BMC Evol Biol 8: 45.
46. Al-Zahery N, Pala M, Battaglia V, Grugni V, Hamod MA, et al. (2011) In
search of the genetic footprints of Sumerians: a survey of Y-chromosome andmtDNA variation in the Marsh Arabs of Iraq. BMC Evol Biol 11: 288.
47. Ottoni C, Martinez-Labarga C, Vitelli L, Scano G, Fabrini E, et al. (2009)
Human mitochondrial DNA variation in Southern Italy. Ann Hum Biol 36:785–811.
48. Derenko M, Malyarchuk B, Grzybowski T, Denisova G, Dambueva I, et al.(2007) Phylogeographic analysis of mitochondrial DNA in northern Asian
populations. Am J Hum Genet 81: 1025–1041.
49. Malyarchuk B, Derenko M, Denisova G, Kravtsova O (2010) Mitogenomicdiversity in Tatars from the Volga-Ural region of Russia. Mol Biol Evol 27:
2220–2226.
50. Schonberg A, Theunert C, Li M, Stoneking M, Nasidze I (2011) High-throughput sequencing of complete human mtDNA genomes from the Caucasus
and West Asia: high diversity and demographic inferences. Eur J Hum Genet 19:
988–994.
51. Behar DM, Van Oven M, Rosset S, Metspalu M, Loogvali E-L, et al. (2012) A
‘‘Copernican’’ reassessment of the human mitochondrial DNA tree from its root.Am J Hum Genet 90: 675–684.
52. Macaulay V, Richards M, Hickey E, Vega E, Cruciani F, et al. (1999) The
emerging tree of West Eurasian mtDNAs: a synthesis of control-region sequencesand RFLPs. Am J Hum Genet 64: 232–249.
53. Macaulay V, Hill C, Achilli A, Rengo C, Clarke D, et al. (2005) Single, rapid
coastal settlement of Asia revealed by analysis of complete mitochondrialgenomes. Science 308: 1034–1036.
54. Hudjashov G, Kivisild T, Underhill PA, Endicott P, Sanchez JJ, et al. (2007)
Revealing the prehistoric settlement of Australia by Y chromosome and mtDNAanalysis. Proc Natl Acad Sci USA 104: 8726–8730.
55. Underhill PA, Myres NM, Rootsi S, Metspalu M, Zhivotovsky LA, et al. (2010)
Separating the post-glacial coancestry of European and Asian Y chromosomeswithin haplogroup R1a. Eur J Hum Genet 18: 479–484.
56. Myres NM, Rootsi S, Lin AA, Jarve M, King RJ, et al. (2011) A major Y-
chromosome haplogroup R1b Holocene era founder effect in Central andWestern Europe. Eur J Hum Genet 19: 95–101.
57. Rootsi S, Magri C, Kivisild T, Benuzzi G, Help H, et al. (2004) Phylogeography
of Y-chromosome haplogroup I reveals distinct domains of prehistoric gene flowin Europe. Am J Hum Genet 75: 128–137.
58. Rootsi S, Zhivotovsky LA, Baldovic M, Kayser M, Kutuev IA, et al. (2007) A
counter-clockwise northern route of the Y-chromosome haplogroup N fromSoutheast Asia towards Europe. Eur J Hum Genet 15: 204–211.
59. Semino O, Magri C, Benuzzi G, Lin AA, Al-Zahery N, et al. (2004) Origin,diffusion, and differentiation of Y-chromosome haplogroups E and J: inferences
on the neolithization of Europe and later migratory events in the Mediterranean
area. Am J Hum Genet 74: 1023–1034.
60. Pericic M, Lauc LB, Klaric IM, Rootsi S, Janicijevic B, et al. (2005) High-
resolution phylogenetic analysis of southeastern Europe traces major episodes of
paternal gene flow among Slavic populations. Mol Biol Evol 22: 1964–1975.
61. Balanovsky O, Dibirova K, Dybo A, Mudrak O, Frolova S, et al. (2011) Parallel
evolution of genes and languages in the Caucasus region. Mol Biol Evol 28:
2905–2920.
Uniparental Genetic Heritage of Belarusians
PLOS ONE | www.plosone.org 13 June 2013 | Volume 8 | Issue 6 | e66499
62. Rootsi S, Myres NM, Lin AA, Jarve M, King RJ, et al. (2012) Distinguishing the
co-ancestries of haplogroup G Y-chromosomes in the populations of Europe and
the Caucasus. Eur J Hum Genet 20: 1275–1282.
63. Alekseeva T (2001) Vostochnye Slaviane. Antropologiya i etnicheskaya istoriya.
Moskva: Nauchnyi Mir. 342 p. (in Russian)
64. Karskii EF (1955) Belorusy: yazyk belorusskogo naroda. Moskva: Izdatelstvo
Akademii nauk SSSR. 517 p. (in Russian)
65. Klimchuk FD (1983) Havorki Zakhodniaha Palessia: fanetychny narys. Minsk:
Navuka i tekhnika. 126 p. (in Belarusian)
66. Yunusbayev B, Metspalu M, Jarve M, Kutuev I, Rootsi S, et al. (2012) The
Caucasus as an asymmetric semipermeable barrier to ancient human migrations.
Mol Biol Evol 29(1): 359–365.
67. Mathew CG (1985) The isolation of high molecular weight eukaryotic DNA.
Methods Mol Biol 2: 31–34.
68. Karafet TM, Mendez FL, Meilerman MB, Underhill PA, Zegura SL, et al.
(2008) New binary polymorphisms reshape and increase resolution of the human
Y chromosomal haplogroup tree. Genome Res 18: 830–838.
69. Torroni A, Rengo C, Guida V, Cruciani F, Sellitto D, et al. (2001) Do the four
clades of the mtDNA haplogroup L2 evolve at different rates? Am J Hum Genet
69: 1348–1356.
70. Rieder MJ, Taylor SL, Tobe VO, Nickerson DA (1998) Automating the
identification of DNA variations using quality-based fluorescence re-sequencing:analysis of the human mitochondrial genome. Nucleic Acids Res 26: 967–973.
71. Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, et al.
(1999) Reanalysis and revision of the Cambridge reference sequence for humanmitochondrial DNA. Nat Genet 23: 147.
72. Bandelt HJ, Forster P, Rohl A (1999) Median-joining networks for inferringintraspecific phylogenies. Mol Biol Evol 16: 37–48.
73. Excoffier L, Lischer HEL (2010) Arlequin suite ver 3.5: a new series of programs
to perform population genetics analyses under Linux and Windows. Mol EcolResour 10: 564–567.
74. Rosenberg MS (2001) PASSAGE: Pattern Analysis, Spatial Statitics andGeographic Exegesis. Arizona State University, Tempe, AZ.
75. Forster P, Harding R, Torroni A, Bandelt HJ (1996) Origin and evolution ofNative American mtDNA variation: a reappraisal. Am J Hum Genet 59: 935–
945.
76. Saillard J, Forster P, Lynnerup N, Bandelt HJ, Nørby S (2000) mtDNA variationamong Greenland Eskimos: the edge of the Beringian expansion. Am J Hum
Genet 67: 718–726.77. Soares P, Ermini L, Thomson N, Mormina M, Rito T, et al. (2009) Correcting
for purifying selection: an improved human mitochondrial molecular clock.
Am J Hum Genet 84: 740–759.
Uniparental Genetic Heritage of Belarusians
PLOS ONE | www.plosone.org 14 June 2013 | Volume 8 | Issue 6 | e66499